PROJECT SUMMARY/ABSTRACT Mitochondria are central to cellular metabolism. They produce energy through oxidative phosphorylation, and participate in the metabolism of amino acids, heme, iron-sulfur clusters, lipids, and other essential cellular metabolites. Alteration in mitochondria function is linked to many diseases, including cancer, diabetes, and numerous neurodegenerative disorders. The majority of the biosynthetic reactions mitochondria participate in occur within the matrix of the mitochondria, which is bounded by two membranes: a permeable outer membrane, and an impermeable inner membrane. Cellular metabolites crucial for these reactions are transported across the inner mitochondrial membrane through nutrient carriers, the largest class of which is the mitochondrial carrier family. Despite their central importance in cellular metabolism, very little is understood about how cells regulate the levels of mitochondrial metabolite transporters to control nutrient compartmentalization. Using yeast as a model system, we recently found that mitochondrial function is intimately linked to the cytoplasmic pool of amino acids. Amino acids are typically stored in the vacuole or lysosome at high levels. However, disruption of efficient storage capability of this organelle leads to rapid mitochondrial dysfunction, likely through metabolic overload. We have now uncovered a mitochondrial quality control pathway that specifically and selectively eliminates mitochondrial carrier proteins in response to changes in cellular amino acid pools. Removal of these proteins occurs through a selective intermediate structure called a mitochondrial derived compartment, or MDC. Our current hypothesis is that sequestration of nutrient carriers by this pathway protects mitochondria in times of cellular metabolic stress. Excitingly, this pathway appears to be just one wing of what we are proposing is a global cellular system for regulation of nutrient transporters to control intracellular amino acid levels. In this application, we lay out a long-term goal of our research program: to ultimately understand how cells regulate metabolite transporters to control cellular nutrient compartmentalization. As a first step in this process, we will focus on three areas outlined in this proposal: 1) understanding how mitochondrial nutrient transporters are selectively removed from the mitochondrial inner membrane for destruction in both yeast and mammals; 2) examining how amino acid levels trigger this pathway, and 3) investigating the coordination of the mitochondrial MDC pathway with similar systems that regulate levels of plasma membrane and lysosomal nutrient transporters. Understanding how intracellular metabolites trigger selective removal and destruction of mitochondrial nutrient carriers will provide important insights into mechanisms of intra-organelle protein sorting, and identify new modes of cellular metabolic regulation.